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Significance

Proteins with a similar structure can have largely different folding properties. Although some fold readily, others can only assume their native structure through the help of chaperone proteins. Partially folded intermediates play a key role in defining those folding differences. However, owing to their transient nature, they are not amenable to the structural investigation. Using a combination of single-molecule mechanics, protein engineering, and crystallography, we identified a stable native-like functional nucleus, which is a critical intermediate for spontaneous folding of the Hsp70 nucleotide-binding domain. Based on our findings, we engineered a chimera turning a homologous but folding-incompetent protein into a spontaneously folding protein that is enzymatically active. Our results have implications for the folding of actin from the same superfamily.

Abstract

The folding pathways of large proteins are complex, with many of them requiring the aid of chaperones and others folding spontaneously. Along the folding pathways, partially folded intermediates are frequently populated; their role in the driving of the folding process is unclear. The structures of these intermediates are generally not amenable to high-resolution structural techniques because of their transient nature. Here we employed single-molecule force measurements to scrutinize the hierarchy of intermediate structures along the folding pathway of the nucleotide binding domain (NBD) of Escherichia coli Hsp70 DnaK. DnaK-NBD is a member of the sugar kinase superfamily that includes Hsp70s and the cytoskeletal protein actin. Using optical tweezers, a stable nucleotide-binding competent en route folding intermediate comprising lobe II residues (183–383) was identified as a critical checkpoint for productive folding. We obtained a structural snapshot of this folding intermediate that shows native-like conformation. To assess the fundamental role of folded lobe II for efficient folding, we turned our attention to yeast mitochondrial NBD, which does not fold without a dedicated chaperone. After replacing the yeast lobe II residues with stable E. coli lobe II, the obtained chimeric protein showed native-like ATPase activity and robust folding into the native state, even in the absence of chaperone. In summary, lobe II is a stable nucleotide-binding competent folding nucleus that is the key to time-efficient folding and possibly resembles a common ancestor domain. Our findings provide a conceptual framework for the folding pathways of other members of this protein superfamily.

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